Phosphoinositide-specific phospholipase C class enzymes are involved in many signaling pathways in which a cellular response (such as proliferation or secretion) is produced consequent to an extracellular stimulus. Distinct isozymes of PLC have been isolated, purified, and/or molecularly cloned from a variety of mammalian tissues. Classified on the basis of their deduced amino acid sequence, the distinct types of PLC isozymes have been identified as PLC-beta, PLC-gamma and PLC-delta (four distinct types of PLC isozymes were originally isolated and identified as PLC-alpha, PLC-beta, PLC-gamma and PLC-delta; the subtypes within the groups were named using Arabic numerals: PLC-β1, PLC-β2, PLC-β3 and PLC-β4 (Rhee, S. G., Suh, P., Ryu, S. & Lee, S. Y., Studies of Inositol Phosphalipid-Specific Phospholipase C, Science, 1989, 244:546-50). PLC-alpha was later determined to be in the PLC-delta class (Rhee S. G. & Choi, K. D., Regulation of Inositol Phospholipid-Specific Phospholipase C Isozymes, Journal of biological Chemistry, 1992, 267:12393-96).
The subtypes differ in their ability to hydrolyze phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) or phosphatidylinositol-4,5-bisphosphate (PIP2) and in their dependence on Ca2+. PIP2 is the main source of phospholipid second messengers and is stored in the inner leaflet of the plasma membrane. PIP2 is derived from PI by a series of kinases. PI is synthesized in the endoplasmic reticulum and is transferred to the inner plasma membrane. PI can also be further phosphorylated by PI-4-kinase, which is membrane associated in most tissues, to give PIP. Finally, PIP can also be phosphorylated by PI(4)P-5-kinases to generate PIP2 (Rhee S. G., Regulation of Phosphoinositide-Specific Phospholipase C, Ann. Rev. Biochem., 2001, 70:221-312, Majerus, Philip W., Inositol Phosphate Biochemistry, Annual Review of Biochemistry, 1992, 61:225-50).
Recruitment and activation of leukocytes are essential components of the inflammatory response. The inflammatory response is primarily controlled by two groups of proteins known as chemokines (e.g. MCP-1 (monocyte chemotactic protein-1)) and cytokines (e.g. tumor necrosis factor-α [TNF-α] or interleukin-1 [IL-1]) (Feng L., Role of Chemokines in Inflammation and Immunoregulation, Immunol. Res., 2000, 21:203-210). Resident tissue cells secrete chemokines and cytokines following tissue injury and/or the detection of the presence of an infectious agent (Gerard C., Rolling B., Chemokines and Disease, Nat. Immunol., 2000, 2:108-115).
Several cytokines (e.g., IL-1 and TNF-α) stimulate vascular endothelial cells to upregulate their expression of adhesion molecules for circulating leukocytes, while chemokines direct the movement of the leukocytes through the endothelial barrier to the site of inflammation and activate such cells once they have migrated into the lesion (Keane M. P., Strieter R. M., Chemokine Signaling in Inflammation, Crit. Care Med., 2000, 28:Suppl 4, N13-N26). Although inflammation plays a critical role in host defense to microorganisms, a poorly-regulated inflammatory response is a primary factor in the pathophysiology of several prevalent autoimmune diseases, has been implicated in the recruitment and activation of mononuclear cells in the synovial membrane in patients with rheumatoid arthritis (RA), and appears to stimulate cartilage and bone destruction. For example, the concentrations of MCP-1 (MCP-1 stimulates the upregulation of adhesion molecules on the surface of monocytes, thereby enhancing their ability to adhere to vascular endothelium, their migratory capacity and their production of superoxide anion, an essential factor in the process of killing phagocytized bacteria (Keane , 2000), MIP-1α, (macrophage inflammatory protein-1α), TNF-α and other chemokines and cytokines are increased in the inflamed joints of patients with RA, with higher levels correlating with increased severity of the disease in both man and experimental animals (Ellingsen T., et al, Plasma MCP-1 is a Marker for Joint Inflammation in Rheumatoid Arthritis, J. Rheumatol., 2001, 28:41-46; Hjelmstrom P., et al, Lymphoid Tissue Homing Chemokines are Expressed in Chronic Inflammation, Am. J. Pathol., 2000, 156:1133-1138; and, Kasama T., et al, Interleukin-10 Expression and Chemokine Regulation During the Evolution of Murine Type ii Collagen-Induced Arthritis, J. Clin. Invest., 1995, 95:2868-2876).
Chemokines also appear to be important mediators in multiple sclerosis (MS). Chemokine concentrations are elevated in the CSF (cerebrospinal fluid) of MS patients, and central nervous system T-cells in MS patients are highly enriched for certain chemokine receptors (Sorensen T. L., et al, Expression of Specific Chemokines and Chemokine Receptors in the Central Nervous System of Multiple Sclerosis Patients, J. Clin. Invest., 1999, 103:807-815). Mice deficient in MCP-1 or CCR2 (the cell-surface receptor for MCP-1) are resistant to the development of experimental autoimmune encephalomyelitis (EAE), a well-characterized animal model of MS (Fife B. T., et al, CC Chemokine Receptor 2 is Critical for Induction of Experimental Autoimmune Encephalomyelitis, J. Exp. Med., 2002, 192:899-905; and Huang D., et al, Absence of Monocyte Chemoattractant-1 in Mice Leads to Decreased Local Macrophage Recruitment and Antigen-Specific T Helper Cell Type 1 Immune Response in Experimental Allergic Encephalomyelitis, J. Exp. Med., 2000, 193:713-725).
Many chemokines (eg interleukin-8 [IL-8]) interact with cell-surface receptors to stimulate PLCβ2 via receptor-linked G-proteins (guanine-nucleotide binding proteins) (Kriz D., et al, Ciba Found, Symp., 1990, 150:112-117). Activation of PLC-β2 by the receptor-linked G-protein catalyzes the hydrolysis of PIP2 to release the second messengers 1,2-diacylglycerol (DAG) and 1,4,5-inositol trisphosphate (IP3). IP3 stimulates intracellular Ca2+ release, while hydrophobic DAG remains in the plasma membrane where it mediates the activation of members of the protein kinase C (“PKC”) family. PLC-β2 is found primarily in hematopoietic cells and can be activated by both the Gα subunits of the Gq class and by the βγ subunits generated by a number of different G-proteins (Park D., et al, Cloning, Sequencing, Expression and Gq-Independent Activation of Phospholipase C-β2, J. Biol. Chem., 1992, 267:16048-16055).
Cotransfection experiments in COS-7 and HEK 293 cells demonstrate clearly that PLC-β2 functions downstream of several chemokine receptors (Wu D., Roles of Phospholipid Signaling in Chemoattractant-Induced Responses, J. Cell Sci., 2000, 113:2935-2940; Huping J., et al, Role of Phospholipase C-β2 in Chemoattractant-Elicited Responses, Proc. Natl. Acad. Sci. (USA), 1997, 94:7971-7975).
For example, experiments with cells expressing transfected receptors for complement component C5a, fMet-Leu-Phe (fMLP) (Sigma, catalog no. F-3506), IL-8 or MCP-1 have shown that each of these receptors activates PLC-β2 through a pertussis toxin (PTx)-sensitive mechanism to release βγ subunits from the Gi class of heterotrimeric G-proteins (Jiang H, et al, Pertussis Toxin-Sensitive Activation of Phospholipase C by the C5a and fMet-Leu-Phe Receptors, J. Biol. Chem., 1996, 271:13430-13434). Additional evidence for the involvement of PLC-β2 in signaling through chemokine receptors comes from experiments in knockout (KO) mice deficient in expression of the PLC-β2 protein. Although hematopoeisis is not affected in these mice, cells from the mice have decreased responsiveness to chemokines as measured by Ca2+ fluxes, generation of inositol phosphates, upregulation of adhesion molecules, phosphorylation of MAP kinases and production of superoxide anion (Wu D., 2000; Huping J., 1997). Surprisingly, however, leukocytes from those mice were reported to have normal or even enhanced chemotactic responses to various chemokines (Park D., 2000; Wu D., 2000; Huping J., 1997). Inhibitors of PLC-β2 enzymatic activity inhibit chemotactic responses to various chemotactic factors, suggesting that a compensatory mechanism may exist in the PLC-β2 KO mice which overcomes the congenital absence of the enzyme to allow normal or enhanced migratory responsiveness to chemokines (Park D., 2000; Wu D., 2000; Huping J., 1997).
References to a number of substituted piperazine and piperidine compounds include those disclosing use as an inhibitor of the NHE1 isoform of the sodium/hydrogen exchanger (Lorrain, J., et al; Pharmacological Profile of SL 591227, A Novel Inhibitor of the Sodium/Hydrogen Exchanger, Brit. J. Pharm., 2000, 131:1188-1194), as platelet aggregation inhibitors (acting as fibrinogen receptor antagonists) (U.S. Pat. No. 5,795,893), as tachykinin receptor antagonists (U.S. Pat. No. 5,607,936), as 5HT2C antagonists (U.S. Pat. No. 5,972,937), as 5HT1D receptor antagonists (U.S. Pat. No. 5,905,080), as enzyme acyl coenzyme A: cholesterol acyltransferase inhibitors (U.S. Pat. No. 5,185,358), as protein isoprenyl tranferase (such as protein farnesyltransferase and protein geranylgeranyltransferase) inhibitors (U.S. Pat. No. 6,310,095), as cardiovascular agents (U.S. Pat. No. 5,547,966) and as antiviral agents (European Patent EP0548798). PCT application WO 93/30322 discloses thiourea compounds for treating AIDS and/or HIV.
The PLC class of enzymes play important roles in inflammatory responses. Therefore, inhibitors of PLC may be useful in treating or ameliorating inflammatory disorders. The present invention provides novel heterocyclyl-substituted anilino compounds which function as PLC inhibitors, thereby providing a means for the treatment and/or amelioration of disorders and conditions mediated by PLC-β2, including inflammatory and related disorders.